| Rad/Rem/Rem2/Gem/Kir (RGK) proteins belong to the Ras superfamily of monomeric GTPases, and potently inhibit voltage dependent calcium (Ca V) channels by binding auxiliary CaVbeta subunits. Because RGK proteins are widely prevalent, and their expression levels differentially regulated in disease, their crosstalk with CaV channels is well-placed to regulate many Cat2+-dependent biological and pathophysiological events. However, the mechanisms underlying ICa ablation by RGK proteins are poorly understood.;Here, we conducted a comprehensive study on the structure-function mechanisms underlying RGK GTPase inhibition of CaV channels using a combination of electrophysiology, flow cytometry, FRET and immunoprecipitation techniques. We found that Rem binds to CaVbeta GK domain with a relatively low affinity, and is unable to inhibit the high-affinity interaction between CaVbeta and the alpha1-subunit domain I-II cytoplasmic loop in pull-down competition assays. These findings are inconsistent with the idea that Rem inhibits ICa by out-competing alpha 1 subunits for binding to Ca Valpha, and rather suggest that Rem inhibits ICa by forming a ternary complex with alpha1/CaVbeta. We discovered that the potent inhibition of recombinant CaV1.2 channels by Rem depends on at least three distinct mechanisms that require different functional conformations of the GTPase. We further found that there were genuine differences among distinct RGK proteins regarding the structure-function mechanisms underlying ICa inhibition.;Finally, based on the new mechanistic insights of RGK GTPases inhibition of ICa, we engineered novel genetically-encoded molecules for inducibly inhibiting CaV channels (GEMIICCs), that have promising applications as key research tools, and potentially, as therapeutics.;Overall, our studies predict new physiological dimensions of the RGK GTPase/CaV channel crosstalk, and suggest original approaches for developing novel CaV channel blockers. |
